Group of nucleic acid molecules salmonella detection, nucleic acids, kit and use

ABSTRACT

The present invention relates to a nucleic acid molecule or molecules and to a process for the detection of bacteria of the Salmonella genus. The invention relates also to a test kit or test kits for carrying out the mentioned detection processes.

[0001] In the foodstuffs and pharmaceutical industries the contaminationof products by pathogenic microorganisms by way of the raw materialsused or during the production or packaging processes poses a majorproblem. Salmonellae are among the most serious pathogens transmitted tohumans through foodstuffs. Since the detection and identification ofSalmonellae by conventional microbiological detection processes is verytime-consuming at least five days are required for the increase inquantity and subsequent serotyping required by legal regulations (LMBG,FDA)—there is a great need for alternative rapid methods.

[0002] In recent years, a number of new methods have been developed forroutine use to detect microorganisms. These include immunologicalprocesses based on the use of polyvalent or monoclonal antibodies andprocesses in which nucleic acid probes are used for detection by meansof hybridisation to organism-specific nucleic acids. Further methodsthat have been described are those processes based on a specific nucleicacid amplification, with or without a subsequent confirmation reactionby nucleic acid hybridisation. Suitable processes for the amplificationof nucleic acids are, for example, polymerase chain reaction [PCR] [U.S.Pat. Nos. 4,683,195; 4,683,202; and 4,965,188], ligase chain reaction[WO Publication 89/09835], “self-sustained sequence replication” [EP 329822], the “transcription based amplification system” [EP 310 229] andthe Qβ RNA-replicase system [U.S. Pat. No. 4,957,858].

[0003] The mentioned nucleic-acid-based processes are so sensitive that,unlike conventional microbiological processes, a lengthy increase inquantity of the microorganism to be detected from the sample to beinvestigated is unnecessary. An investigation of the presence or absenceof, for example, Salmonellae is therefore generally concluded within oneworking day when using the mentioned nucleic-acid-based processes.

[0004] Some nucleic acid sequences for detecting Salmonellae bypolymerase chain reaction are known. A disadvantage is, however, thatwhen using those nucleic acid sequences as primers in the polymerasechain reaction false positive results [WO 95/33854] or false negativeresults [WO 92/01056; WO 95/00664; WO 92/01056; WO 93/04202] occur. Inother cases, only an insufficient number of strains of all 7 Salmonellaesubspecies have been studied [WO 92/08805; WO 94/25597; DE 4337295], sothat thus far it is unclear whether the nucleic acid sequences inquestion are suitable for detecting all Salmonella strains.

[0005] An advantage of, for example, the primers and probes described inInternational Patent Application WO 95/00664 is that they allow thehighly selective detection of bacteria of the Salmonella genus withoutthe occurrence of false positive results. A disadvantage when using theoligonucleotides according to WO 95/00664 in amplification processessuch as polymerase chain reaction is, however, the fact that none of thedescribed primer pairs enable detection of all the representatives ofthe 7 Salmonella subspecies. For example, when using the primersST11/ST15, a number of representatives of subspecies IIIa (subsp.arizonae) are not detected, and when using the primers ST11/ST14 anumber of representatives of subspecies I (subsp. enterica Serovar.Blockley) and of subspecies IIIa (subsp. arizonae) are not detected.

[0006] An aim of the invention described herein was to optimise thedetection processes described in WO 95/00664 by finding nucleic acidsequences the use of which as primers and/or probes ensures as completedetection as possible of all the representatives of the Salmonellagenus.

[0007] According to an embodiment, the problem underlying the inventionis solved by a set of nucleic acid molecules by means of which, in aprocess for the detection of representatives of Salmonella entericasubsp. enterica, salamae, arizonae, diarizonae, houtenae, bongori andindica, all the representatives of those subspecies can be detected, theset being obtainable by

[0008] (a) obtaining or deriving a first nucleic acid molecule (nucleicacid molecule 1) in a manner known per se using a nucleic acid isolateof a representative of one of the mentioned Salmonella entericasubspecies, which first nucleic acid molecule is specifically suitableas primer or probe for the detection of that representative or offurther or all representatives of that one Salmonella entericasubspecies and possibly also of representatives of further Salmonellaenterica subspecies,

[0009] (b) obtaining or deriving a second nucleic acid molecule (nucleicacid molecule 2) in a manner known per se using a nucleic acid isolateof a different representative of one of the mentioned Salmonellaenterica subspecies, which second nucleic acid molecule is specificallysuitable as primer or probe for the detection of that representative orof further or all representatives of that different Salmonella entericasubspecies and possibly also of representatives of others of thementioned Salmonella enterica subspecies, and

[0010] (c) unless it is already possible to detect all therepresentatives of the mentioned Salmonella enterica subspecies usingthe nucleic acid molecules obtainable according to (a) and (b),continuing to obtain or derive nucleic acid molecules according to (a)and/or (b) until all the representatives of the mentioned Salmonellaenterica subspecies can be detected using the obtained or derived set ofnucleic acid molecules.

[0011] A derived nucleic acid molecule may be a nucleic acid moleculethat can be hybridised with the obtained nucleic acid molecule and thatpreferably has the same number of bases, possible hybridisationconditions being:

temperature ≧25° C. and 1M NaCl concentration.

[0012] A derived nucleic acid molecule may be, for example, a nucleicacid molecule the sequence of which has been determined by computerdesign and that has subsequently been manufactured and obtained bychemical synthesis.

[0013] The solution to the problem underlying the invention can also bedescribed as the provision of one or more nucleic acid molecule(s) Y (Z,. . . ), that(those) nucleic acid molecule(s) being characterised inthat the use of that(those) nucleic acid molecule(s)—in addition to theuse of a nucleic acid molecule (X)—in a process for the detection ofbacteria of the Salmonella genus enables the detection also ofSalmonella strains or Salmonella isolates that cannot be detected or canbe detected only with relatively low sensitivity using the nucleic acidmolecule (X).

[0014] The set of nucleic acid molecules according to the invention canbe characterised in that the nucleic acid isolates comprise or arephylogenetically conserved base sequences or regions of those basesequences. For the term “phylogenetically conserved base sequence”, see,for example, WO 95/00664 or Herder's Lexikon der Biochemie undMolekularbiologie, supplemented 1995, page 132, spectrum, production,etc..

[0015] The set of nucleic acid molecules according to the invention canbe characterised in that the individual nucleic acid molecules or someof the nucleic acid molecules hybridise to

[0016] (i) different phylogenetically conserved base sequences, or

[0017] (ii) one and the same phylogenetically conserved base sequence atnon-overlapping sequence regions, or

[0018] (iii) one and the same phylogenetically conserved base sequenceat overlapping sequence regions.

[0019] The set of nucleic acid molecules according to the invention or aset of nucleic acid molecules according to the invention by means ofwhich, in a process for the detection of representatives of Salmonellaenterica subsp. enterica, salamae, arizonae, diarizonae, houtenae,bongori and indica, all the representatives of those subspecies can bedetected, can be characterised in that the set for an individual nucleicacid molecule, for a number of its individual nucleic acid molecules orfor each of its individual nucleic acid molecules in each case comprisesat least one further nucleic acid molecule that, in a region of at least10 successive nucleotides of their nucleotide chain, corresponds to lessthan 100% but to at least 80% of the base sequence.

[0020] Such a set of nucleic acid molecules according to the inventioncan be characterised in that the set for an individual nucleic acidmolecule, for a number of its individual nucleic acid molecules or foreach of its individual nucleic acid molecules in each case comprises atleast one further nucleic acid molecule that, in a region of at least 10successive nucleotides of their nucleotide chain, differs from the otheror further nucleic acid molecule in precisely one base position.

[0021] A set of nucleic acid molecules according to the invention can becharacterised in that it comprises one or more, but not exclusively,nucleic acid molecules that are fragments of the SEQ ID NO 1 accordingto WO 95/00664 or of its complementary sequence.

[0022] A set of nucleic acid molecules according to the invention canalso be characterised in that the individual nucleic acid moleculeshybridise to the same strand of nucleic acid isolates of representativesof Salmonella enterica subspecies that are being subjected to theprocess for their detection.

[0023] The problem underlying the invention is also solved by a nucleicacid molecule that belongs to a set of nucleic acid molecules accordingto the invention or that can be used for such a set, the nucleic acidmolecule being characterised in that, in a region of at least 10successive nucleotides of its nucleotide chain, the sequence of thenucleic acid molecule corresponds exactly to a sequence region of atleast one representative of the mentioned Salmonella entericasubspecies, the sequence region comprising or being a phylogeneticallyconserved base sequence or a region of that base sequence.

[0024] Such a nucleic acid molecule according to the invention can becharacterised in that, in a region of at least 10 successive nucleotidesof its nucleotide chain, it is 100% or at least 80% identical to acorresponding number of successive nucleotides of one or more of thefollowing sequences or their complementary sequences:ATGGATCAGAATACGCCCCG SEQ ID NO:1 ATGGATCAGAATACACCCCG SEQ ID NO:2CAGAATACGCCCCGTTCGGC SEQ ID NO:3 CAGAATACACCCCGTTCGGC SEQ ID NO:4CAGAATACGCCCCGTTCAGC SEQ ID NO:5 CAACCTAACTTCTGCGCCAG SEQ ID NO:6CAACCTAACTTCTGCACCAG SEQ ID NO:7 CAACCTAACCTCTGCGCCAG SEQ ID NO:8CAACCTAACTTCTGCGCCAG SEQ ID NO:9

[0025] The problem underlying the invention is also solved by a nucleicacid molecule characterised in that, in respect of its sequence, it ishomologous to an above-characterised nucleic acid molecule according tothe invention and, in at least 10 successive nucleotides of itsnucleotide chain,

[0026] (i) is identical to an above-characterised nucleic acid moleculeaccording to the invention, or

[0027] (ii) differs from an above-characterised nucleic acid moleculeaccording to the invention in not more than one nucleotide, or

[0028] (iii) differs from an above-characterised nucleic acid moleculeaccording to the invention in not more than two nucleotides.

[0029] A nucleic acid molecule according to the invention can becharacterised in that it is from 10 to 250 nucleotides long andpreferably from 15 to 30 nucleotides long.

[0030] A nucleic acid molecule according to the invention can also becharacterised in that it is single-stranded or has a complementarystrand.

[0031] A nucleic acid molecule according to the invention can also becharacterised in that it is present

[0032] (i) as DNA, or

[0033] (ii) as RNA corresponding to (i), or

[0034] (iii) as PNA, the nucleic acid molecule where appropriate havingbeen modified or labelled in a manner known per se for analyticaldetection processes, especially detection processes based onhybridisation and/or amplification.

[0035] A nucleic acid molecule according to the invention can also becharacterised in that it is a modified or labelled nucleic acid moleculein which up to 20% of the nucleotides of at least 10 successivenucleotides of its nucleotide chain are building blocks known per se asprobes and/or primers, especially nucleotides that do not occurnaturally in bacteria.

[0036] A nucleic acid molecule according to the invention can also becharacterised in that it is a modified or labelled or additionallymodified or labelled nucleic acid molecule that comprises, in a mannerknown per se for analytical detection processes, one or more radioactivegroups, coloured groups, fluorescent groups, groups for immobilisationon a solid phase, groups for an indirect or direct reaction, especiallyfor an enzymatic reaction, preferably using antibodies, antigens,enzymes and/or substances having an affinity for enzymes or enzymecomplexes, and/or other modifying or modified groups ofnucleic-acid-like structure that are known per se.

[0037] The problem underlying the invention is also solved by a kit foranalytical detection processes, especially for the detection of bacteriaof the Salmonella genus, that kit being characterised by

[0038] (i) a set of nucleic acid molecules according to the invention,or

[0039] (ii) one or more nucleic acid molecules according to theinvention.

[0040] A kit according to the invention can thus comprise a set ofnucleic acid molecules according to the invention or one or more nucleicacid molecules according to the invention, there additionally beingprovided the other customary components for nucleic acid hybridisationsor nucleic acid amplifications, for example a polymerase, a reversetranscriptase, a ligase or an RNA-polymerase, see, for example, WO95/00664.

[0041] A set of nucleic acid molecules of the kit according to theinvention will preferably be produced synthetically in at least twoseparate synthesis batches. The kit according to the inventionpreferably does not comprise any degenerate nucleic acid molecules.

[0042] Finally, the problem underlying the invention is solved by theuse of a set of nucleic acid molecules according to the invention or ofa kit according to the invention to detect the presence or absence ofbacteria belonging to a group of bacteria of the Salmonella genus,especially of representatives of the above-mentioned Salmonella entericasub-species.

[0043] For the use according to the invention, nucleic acidhybridisation and/or nucleic acid amplification can be carried out.

[0044] As nucleic acid amplification, there can be carried out apolymerase chain reaction (PCR).

[0045] For the use according to the invention, differences between thegenomic DNA and/or RNA of the bacteria to be detected and of thebacteria that are not to be detected can be determined at at least onenucleotide position in the region of a nucleic acid molecule accordingto the invention and representatives of a group of bacteria of theSalmonella genus can be detected, especially representatives of thementioned Salmonella enterica subspecies.

[0046] To detect Salmonellae by means of nucleic acid hybridisation oramplification, Salmonella-specific oligonucleotides are used.Salmonella-specific oligonucleotides are nucleic acid molecules, from 10to 250 bases (preferably from 15 to 30 bases) long, the base sequence ofwhich is characteristic for Salmonellae: when using sucholigonucleotides as primers or probes—with suitable reactionconditions—hybridisation/amplification takes place only when DNA of theSalmonellae to be detected is present in the test sample, but not whenDNA of other bacteria is present.

[0047] As described below, given certain prerequisites non-specificoligonucleotides may also be used as primers or probes. Sucholigonucleotides enable hybridisation and/or amplification not only whenSalmonella-DNA is present in the sample but also in the presence of DNAof a bacterium or of a number of bacteria not belonging to theSalmonella genus.

[0048] Since, in highly conserved gene regions, substitutions (e.g.point mutations) in the DNA can occur even in the case of very closelyrelated bacteria, comprehensive DNA sequencing or specificity tests(e.g. by carrying out PCR) are necessary to select suitableoligonucleotides. This applies equally to bacteria to be detected (i.e.Salmonellae) and to bacteria that are not to be detected (i.e. bacterianot belonging to the Salmonella genus).

[0049] To detect Salmonellae, firstly nucleic acids, preferably genomicDNA, are released from the cells contained in a sample or bacterialculture to be investigated. By means of nucleic acid hybridisation, thedirect detection of Salmonella nucleic acids in the sample to beinvestigated can then be effected using the Salmonella-specificoligonucleotides according to the invention as probe. Various processesknown to the person skilled in the art are suitable for that purpose,such as, for example, “Southern blot” or “dot blot”.

[0050] Preference is given, however, above all on account of therelatively high sensitivity, to indirect detection in which the DNA/RNAsequences sought are firstly amplified by means of the above-mentionedprocesses for amplifying nucleic acids, preferably PCR. Theamplification of DNA/RNA is effected by using Salmonella-specificoligonucleotides. In that process specific amplification products areformed only when Salmonella-DNA/RNA is present in the sample to beinvestigated. The specificity of the detection process can be increasedby a subsequent detection reaction using Salmonella-specificoligonucleotides as probes. It is also possible to use non-specificoligonucleotides as probes.

[0051] Alternatively, amplification can also be carried out in thepresence of one or more non-specific oligonucleotides, so that possiblyalso DNA/RNA of other microorganisms that are not to be detected may beamplified. Such an amplification process is generally less specific andshould therefore be backed up by a subsequent detection reaction usingSalmonella-specific oligonucleotides as probe.

[0052] Various processes by which amplification products formed in theindirect processes can be detected are known to the person skilled inthe art. These include, inter alia, visualisation by means of gelelectrophoresis, the hybridisation of probes on immobilised reactionproducts [coupled to nylon or nitrocellulose filters (“Southern blots”)or, for example, on beads or microtitre plates] and the hybridisation ofthe reaction products on immobilised probes (e.g. “reverse dot blots” orbeads or microtitre plates coupled with probes).

[0053] A large number of different variants have been described by meansof which the described Salmonella-specific or non-specificoligonucleotides for use as probes and/or primers in direct or indirectdetection processes can be labelled or modified. They may comprise, forexample, radioactive, coloured or fluorescent groups or groups thatenable immobilisation on a solid phase or groups that have been modifiedor that modify in some other way, such as, for example, antibodies,antigens, enzymes or other substances having an affinity for enzymes orenzyme complexes. Probes and primers may be either naturally occurringor synthetically produced double- or single-stranded DNA or RNA ormodified forms of DNA or RNA, such as, for example, PNA (in thosemolecules the sugar units have been replaced by amino acids orpeptides). Individual nucleotides or a number of nucleotides of theprobes or primers may be replaced by analogous building blocks (such as,for example, nucleotides that do not naturally occur in the targetnucleic acid). In the case of the above-mentioned indirect detectionprocesses, the detection can be carried out also by means of aninternally labelled amplification product. That can be effected, forexample, by the integration of modified nucleoside triphosphates (e.g.coupled with digoxygenin or fluorescein) during the amplificationreaction.

[0054] Suitable Salmonella-specific oligonucleotides according to theinvention are nucleic acids, preferably from 15 to 30 bases long, thatcorrespond, at least in a 10 base long sequence, to sequences 1 to 10 orto their complementary sequences. Relatively small differences (1 or 2bases) in that 10 base long sequence are possible without loss of therequisite specificity in the amplification and/or hybridisation. Theperson skilled in the art will know that in the case of such relativelysmall differences the reaction conditions need to be alteredaccordingly.

[0055] In order to enable complete detection of all the Salmonellastrains using the DNA region outlined in WO 95/00664, comprehensive DNAsequence analyses were necessary. The sequence of that DNA region wasdetermined from 37 selected Salmonella strains of all 7 subspecies (formost of the strains, the sequence of the DNA region between primers ST15and ST11; this corresponds to position 1275 to 1654 of SEQ ID NO: 1 inWO 95/00664). Possible experimental procedures will be known to theperson skilled in the art and will not be described here in detail; abrief summary of the results will be given. DNA of the selectedSalmonella strains was prepared by standard procedures and the relevantregion was amplified by PCR and subsequently sequenced. In the PCR andthe subsequent sequencing, the following primers were used for most ofthe Salmonella strains:

[0056] ST11: AGCCAACCATTGCTAAATTGGCGCA (see claim 3, WO 95/00664)

[0057] ST15: GGTAGAAATTCCCAGCGGGTACTG (see claim 3, WO 95/00664).

[0058] Since, however, no amplification, or only insufficientamplification, occurred with that primer pair with a number of strainsof subspecies IIIa, IV, V and VI, in those cases the following primerswere used for the PCR and sequencing:

[0059] ST11: AGCCAACCATTGCTAAATTGGCGCA (see claim 3, WO 95/00664)

[0060] ST14: TTTGCGACTATCAGGTTACCGTGG (see claim 3, WO 95/00664).

[0061] A comparison of the DNA sequences of all 37 Salmonella strainsshowed that while it was as a whole a conserved DNA region, the degreeof conservation appeared at first glance to have only limitedsuitability for deriving Salmonella-specific oligonucleotides. Even inthe most highly conserved regions, base substitutions were observed insome of the sequenced strains. Interestingly, it was found that many ofthe base substitutions occur only within a subgroup and that thesubstitutions are moreover generally conserved within that subgroup.This suggested the possibility of using more than two primers in the PCRin order to enable amplification also of those variants in which one ormore base substitutions are present in the region of the primer bindingsites. As the person skilled in the art will know, for that purposethere are customarily used degenerate primers or primers havingdeoxyinosin at the variable sites. A number of degenerateoligonucleotides that were potentially suitable as primers for thedetection of all Salmonella enterica subspecies were therefore deducedfrom the above-mentioned sequence comparison. It was found, however,that those degenerate primers have only limited suitability for PCRdetection since they result in an increase in the occurrence ofnon-specific reaction products, especially in the case of sequenceregions of high complexity. Since the sensitivity of the PCR detectiongenerally suffers from the occurrence of such non-specific reactionproducts, a different procedure was tried. “Complementing” primers wereused in the PCR. In contrast to degenerate primers, in which all thepossible combinations of the individual base substitutions arerepresented in the primer mixture (number of primers=2^(x)×3^(Y)×4^(z)where x, y and z are the number of positions at which two, three or fourdifferent bases are observed in the region of the primer binding site),in such complementing primers only the actually occurring sequences arepresent. The advantage over degenerate primers lies in the lessercomplexity of the primer mixture according to the invention, as a resultof which the probability that non-specific amplification products willbe formed is markedly reduced. As has been shown in a number ofexperiments, this is especially advantageous in PCR detection usingsamples having a high content of “non-specific” DNA (DNA that does notcome from bacteria to be detected) since, otherwise, the sensitivity ofthe detection may be radically reduced.

[0062] A major advantage when using complementingoligonucleotides/primers lies in the possibility of optimising existingdetection processes. For example, it is possible that individual falsenegative results can be eliminated by additionally using in the PCRand/or hybridisation reaction oligonucleotides comprising the sequenceof the previously undetected strains.

[0063] The DNA sequence comparison yielded a number of relatively shortDNA regions that appeared to be potentially suitable for the strategydescribed (use of in total ≧3 primers in the PCR) for optimising theSalmonella detection process. The following Example is given by way ofclarification.

EXAMPLE 1 Detection of Salmonella Strains of all 7 Subspecies byPolymerase Chain Reaction

[0064] The following 3 sections of the sequenced DNA region are to serveas examples for the sequence variations observed:

[0065] Section I (position 1336 to 1355 of SEQ ID NO: 1 in WO 95/00664)ATGGATCAGAATACGCCCCG SEQ ID NO:1 ATGGATCAGAATACACCCCG SEQ ID NO:2

[0066] Section II (position 1342 to 1361 of SEQ ID NO: 1 in WO 95/00664)CAGAATACGCCCCGTTCGGC SEQ ID NO:3 CAGAATACACCCCGTTCGGC SEQ ID NO:4CAGAATACGCCCCGTTCAGC SEQ ID NO:5

[0067] Section III (complementary to position 1483 to 1502 of SEQ ID NO:1 in WO 95/00664) CAACCTAACTTCTGCGCCAG SEQ ID NO:6 CAACCTAACTTCTGCACCAGSEQ ID NO:7 CAACCTAACCTCTGCGCCAG SEQ ID NO:8 CAACCTAACTTCTGCGGCAG SEQ IDNO:9 CAACCTAACTTCTGCGGCAG SEQ ID NO:10

[0068] To test whether those sequence sections are suitable fordetecting all the Salmonella strains of the 7 subspecies, theoligonucleotides Sa 1 to 10 were used in the PCR in the followingcombinations:

[0069] Primer combination 1: Sa1/Sa2 (each in a final concentration of0.2 μM) Sa6/Sa7/Sa8/Sa9/Sa10 (each in a final concentration of 0.08 μM)

[0070] Primer combination 2: Sa3/Sa4/Sa5 (each in a final concentrationof 0.13 μM) Sa6/Sa7/Sa8/Sa9/Sa10 (each in a final concentration of 0.08μM)

[0071] DNA was isolated by standard processes from pure cultures of theSalmonella strains listed in Table 1a. Approximately from 10 to 100 ngof each of those DNA preparations was then used in the PCR in thepresence of primer combination 1 or primer combination 2, 200 μM ofdNTP's (Boehringer Mannheim), 1.5 mM MgCl₂, 16 mM (NH₄)₂SO₄, 67 mMTris/HCl (pH 8.8), 0.01% Tween 20 and 0.03 U/μl Taq-polymerase(Biomaster). The PCR was carried out in a Perkin-Elmer 9600 thermocyclerhaving the following thermoprofile: initial denaturing 95° C.  5 minamplification (35 cycles) 95° C. 30 sec 63° C. 90 sec final synthesis72° C.  5 min

[0072] After the end of the PCR reaction, the amplification productswere separated by means of agarose gel electrophoresis and visualised bystaining with ethidium bromide. The expected product of 167 bp length(primer combination 1) or of 161 bp length (primer combination 2) wasobserved in all cases in which DNA of strains of the Salmonella genuswas present (compare Table 1a), but not in the presence of DNA of othertested bacteria (compare Table 1b). After the end of the run, the DNAcontained in the gels was transferred by standard methods to nylonfilters and hybridised with the oligonucleotide ST14(TTTGCGACTATCAGGTTACCGTGG (see claim 3, WO 95/00664)) labelled at the 5′end with digoxygenin to test the base specificity especiallysensitively. Hybridisation was effected in 5×SSC, 2% blocking reagent,0.1% lauryl sarcosine, 0.02% SDS and 5 pmol/ml of probe for 4 hours at60° C. Washing was carried out in 2×SSC, 0.1% SDS for 2×15 minutes at60° C. Detection was carried out according to standard methods usinganti-digoxygenin/alkaline phosphatase conjugates in the presence of5-bromo-4-chloro-3-indolyl phosphate and 4-nitro-blue tetrazoliumchloride (Boehringer Mannheim).

[0073] A band was observed on the filters only in those cases in which aband had previously been visible on the agarose gel (see Table 1a).Thus, the presence of the 296 tested Salmonella strains from each of the7 subspecies was detected both by PCR and by hybridisation. A positivesignal was obtained for each of those strains with primer combination 1,primer combination 2 and, in the subsequent confirmation reaction, byhybridisation with the probe ST14. By contrast, none of the testedbacterial strains not belonging to that genus was detected using thissystem. TABLE 1a Positive Salmonella strains in PCR amplification usingthe two primer combinations 1 or 2 and in the subsequent hybridisationusing the oligonucleotide ST14. No. Subspecies Serogroup Serovar. S.enterica B Abony subsp. Abortusovis Enterica Africana Agona Agona,lactose + Arechavaleta Brandenburg Bredeney Chester Coeln Derby O: 5 −Duisburg Duisburg, monophase Heidelberg Heidelberg, O5 − I 4, 12: d: − I4, 12: −: − I 9, 12: 1, v: − Indiana Kiambu Kunduchi Paratyphi BParatyphi B H 1, 2 negative Paratyphi B O5 − Reading Saintpaul O5 −Saintpaul Sandiego Schleisheim Schwarzengrund Stanley StanleyvilleTyphimurium Typhimurium 4: i: 1, 2 (O: 5−) Typhimurium O: 5 −Typhimurium, TA 1535 Typhimurium, TA 1537 Typhimurium, TA 1538Typhimurium, TA 97 Typhimurium, TA 98 Typhimurium, TA 100 C₁Augustenborg Bareilly Braenderup Choleraesuis Choleraesuis var. DecaturCholeraesuis var. Kunzendorf Colindale Concord Infantis Isangi LilleLivingstone Mbandaka Mikawasima Montevideo Ohio Oranienburg OsloRichmond (2 isolates tested) Rissen Singapore Tennessee Thompson (2isolates tested) Virchow I 6, 7: −: − (2 isolates tested) C₂ C₃ Albany(2 isolates tested) Altona Apeyeme Bardo Blockley BovismorbificansCharlottenburg Cottbus Emek Ferruch Glostrup Goldcoast Haardt HadarKentucky Litchfield Manchester Manhattan Molade Munchen Newport TakoradiI 6, 8: −: − I 8, 20: −: − D₁ Dublin Durban Enteritidis Enteritidisplasmid phage 37MD EnteritidiS PT 4/6 Enteritidis, phage GallinarumGallinarum-Pullorum Israel Javiana Kapemba Napoli Panama Pullorum I 9,12: −: − D₂ Plymoth E₁ Amager Amsterdam O: −, 15+, 34+ Anatum Anatum O15+ Anatum O: 10 −, O: 15+ Birmingham Butantan Falkensee Give LexingtonLondon Meleagridis Munster Munster, O: 10 −, 15+ Orion Orion O: 10 −,15+ 34+ Sinstorf Stockholm Uganda (2 isolates tested) Vejle (2 isolatestested) Weltevreden Westhampton Zanzibar I 3, 10: −: 6 (monophase) I 10:−: 1, 6 E₄ Abaetuba Aberdeen Cannstatt Llandoff Senftenberg, delayedLac. + I 1, 3, 19,: −: − F Chandans (2 isolates tested) Kisarawe KrefeldLiverpool Rubislaw Senftenberg Solt Telashomer G Grumpensis HavanaIdikan Kedougou Poona Putten Worthington I 13, 23,: − H Caracas CharityLindern Onderstepoort Sundsvall Gaminara Hvittingfoss Malstatt Saphra JBonames K Cerro L Minnesota (2 isolates tested) Ruiru M Cotham GuildfordIlala Loeben Mundonobo Nima Patience Pomona Taunton Wedding N AquaMorningside Urbana O Adelaide Alachua Ealing Haga Monschaui P LansingRoan (2 isolates tested) Shettfield Q Kokomelemle R Johannesburg SWaycross (2 isolates tested) T Waral U Thetford V Koketime (2 isolatestested) Lawra W Suelldorf X I 47, z₄, z₂₃: (monophase) Mountpleasant S.enterica B II 4, 12: a: − subsp. C₁ II 6, 7: d: 1, 7 salamae F II 11: g,m, s, t: z₃₉ I II 16: g, m, s, t: − J II 17: c: z₃₉ II 17: b: e, n, x,z₁₅ L II 21: z₁₀: − P II 38: d: 1, 5 R II 1, 40: z₄₂: 1, 5, 7 S II 41:z₁₀, 1, 2 T II 42: r: − (3 isolates tested) X II 47: a: 1, 5 (2 isolatestested) II 47: b: 1, 5 (2 isolates tested) II 47: b: z₆ Z II 50: b: z₆(5 isolates tested) O: 58 II 58: 1, z₁₃ , z₂₈: z₆ S. enterica J IIIa 17:z₄, z_(32: −) subsp. K IIIa 18: z₄, z₂₃: − arizonae P IIIa 38: 1, v: − RIIIa 40: z₄, z₂₄: − S IIIa 41: z₄, z₂₃: − U IIIa 43: g, z₅₁: − V IIIa44: z₄, z₃₂: − IIIa 44: z₄₁, z₂₃: − Y IIIa 48: (1): − IIIa 48: g, z₅₁ :− IIIa 48: z₃₆: − IIIa 48: z₄, z₂₃: − Z IIIa 50: z₄, z₂₄: − O: 51 IIIa51: z₄, z₂₃: − IIIa 51: g, z₅₁: − O: 53 IIIa 53: z₄, z₂₃, z₃₂: − IIIa53: z₂₉: − O: 62 IIIa 62: z₃₆: − O: 63 IIIa 63: g, z₅₁: − S. enterica D₁IIIb 1, 9, 12: y: z₃₉ subsp. I IIIb 16: k: − diarizonae J IIIb 17: z₁₀,e, n, x, z₁₅ O IIIb 35: k: e, n, z₁₅ P IIIb 38: 1, v: z₅₃ IIIb 38: 1, v:z₅₄ T IIIb 42: k: z₃₅ X IIIb 47: b: z₆ IIIb 47: k: z₃₅ IIIb 47: r: z₅₃IIIb 47: −: − Y IIIb 48: (k): z₅₃ Z IIIb 50: k: z IIIb 50: r: z O: 53IIIb 53: 1, k: z O: 60 IIIb 60: z₅₂: z₅₃ O: 61 IIIb 61: 1: z IIIb 61: 1,v: 1, 5, 7 IIIb 61: 1, v: 1, 5, 7: (z₅₇) IIIb 61: r: z₅₃ S. enterica FIV 11: z₄, z₂₃: − subsp. I IV 16: z₄, z₃₂: − houtenae I IV 16: z₄, z₃₂:− J IV 17: z₂₉: − K IV 18: z₃₆, z₃₈: − L IV 21: g, z₅₁: − U IV 43: z₄,z₂₃: − IV 43: z₄, z₃₂: − V TV 44: z₄, z₃₂: − Y IV 48: z₂₉: − Z IV 50:z₄, z₂₃: − S. enterica R V 40: z₃₅: − subsp. V 40: z₈₁: − bongori V V44: d: − V 44 : z₃₉: − Y V 48 : z₃₅: − S. enterica S VI 41: b: 1, 7subsp. W VI 45: a: e, n, x, (z₁₇) indica Y VI 48: z₁₀: 1, 5 VI 48: z₄₁:− VI 1, v: z₆₇

[0074] TABLE 1b Negative strains of non-Salmonella species in PCRamplification using the two primer combinations 1 or 2 and thesubsequent hybridisation using the oligonucleotide ST 14 No. SpeciesOrigin Bacillus subtilis ATCC 6051 Citrobacter freundii DSM 30040Clostridium bifermentans DSM 630 Enterobacter agglomerans IfGB 0202Enterobacter cloacae DSM 30054 Erwinia carotovora DSM 30168 Escherichiacoli ATCC 8739 Hafnia alvei IfGB 0101 Klebsiella oxytoca DSM 5175Klebsiella pneumoniae ATCC 13883 Klebsiella oxytoca DSM 5175Lactobacillus spec. ATCC 20182 Listeria monocytogenes ATCC 19118Pediococcus domnatus IfGB 0101 Proteus vulgaris DSM 2041 Pseudomonsasfluorescens DSM 6290 Serratia marcescens IfGB 0101 Shigella flexneri DSM4782 Staphylococcus aureus ATCC 6538 Yersinia enterocolitica DSM 4780

1. Set of nucleic acid molecules by means of which, in a process for thedetection of representatives of Salmonella enterica subsp. enterica,salamae, arizonae, diarizonae, houtenae, bongori and indica, all therepresentatives of those subspecies can be detected, which set ofnucleic acid molecules is obtainable by (a) obtaining or deriving afirst nucleic acid molecule (nucleic acid molecule 1) in a manner knownper se using a nucleic acid isolate of a representative of one of thementioned Salmonella enterica subspecies, which first nucleic acidmolecule is specifically suitable as primer or probe for the detectionof that representative or of further or all representatives of that oneSalmonella enterica subspecies and possibly also of representatives offurther Salmonella enterica subspecies, (b) obtaining or deriving asecond nucleic acid molecule (nucleic acid molecule 2) in a manner knownper se using a nucleic acid isolate of a different representative of oneof the mentioned Salmonella enterica subspecies, which second nucleicacid molecule is specifically suitable as primer or probe for thedetection of that representative or of further or all representatives ofthat different Salmonella enterica subspecies and possibly also ofrepresentatives of others of the mentioned Salmonella entericasubspecies, and (c) unless it is already possible to detect all therepresentatives of the mentioned Salmonella enterica subspecies usingthe nucleic acid molecules obtainable or derivable according to (a) and(b), continuing to obtain or derive nucleic acid molecules according to(a) and/or (b) until all the representatives of the mentioned Salmonellaenterica subspecies can be detected using the obtained or derived set ofnucleic acid molecules, wherein (d) the nucleic acid isolates compriseor are phylogenetically conserved base sequences or regions of thosebase sequences, wherein (e) the set of nucleic acid molecules startingfrom the nucleic acid molecules that are obtainable or derivableaccording to steps (a) to (d) were produced synthetically and in atleast two separate synthesis batches, wherein (f) the individual nucleicacid molecules or some of the nucleic acid molecules hybridise to (i)different phylogenetically conserved base sequences, or (ii) one and thesame phylogenetically conserved base sequence at non-overlappingsequence regions, or (iii) one and the same phylogenetically conservedbase sequence at overlapping sequence regions, and wherein (g) the setfor an individual nucleic acid molecule, for a number of its individualnucleic acid molecules or for each of its individual nucleic acidmolecules in each case comprises at least one further nucleic acidmolecule that, in a region of at least 10 successive nucleotides oftheir nucleotide chains, corresponds to less than 100% but to at least80% of the base sequence.
 2. Set of nucleic acid molecules by means ofwhich, in a process for the detection of representatives of Salmonellaenterica subsp. enterica, salamae, arizonae, diarizonae, houtenae,bongori and indica, all the representatives of those subspecies can bedetected, which set of nucleic acid molecules is obtainable by (a)obtaining or deriving a first nucleic acid molecule (nucleic acidmolecule 1) in a manner known per se using a nucleic acid isolate of arepresentative of one of the mentioned Salmonella enterica subspecies,which first nucleic acid molecule is specifically suitable as primer orprobe for the detection of that representative or of further or allrepresentatives of that one Salmonella enterica subspecies and possiblyalso of representatives of further Salmonella enterica subspecies, (b)obtaining or deriving a second nucleic acid molecule (nucleic acidmolecule 2) in a manner known per se using a nucleic acid isolate of adifferent representative of one of the mentioned Salmonella entericasubspecies, which second nucleic acid molecule is specifically suitableas primer or probe for the detection of that representative or offurther or all representatives of that different Salmonella entericasubspecies and possibly also of representatives of others of thementioned Salmonella enterica subspecies, and (c) unless it is alreadypossible to detect all the representatives of the mentioned Salmonellaenterica subspecies using the nucleic acid molecules obtainable orderivable according to (a) and (b), continuing to obtain or derivenucleic acid molecules according to (a) and/or (b) until all therepresentatives of the mentioned Salmonella enterica subspecies can bedetected using the obtained or derived set of nucleic acid molecules,wherein (d) the nucleic acid isolates comprise or are phylogeneticallyconserved base sequences or regions of those base sequences, wherein (e)the individual nucleic acid molecules or some of the nucleic acidmolecules hybridise to (i) different phylogenetically conserved basesequences, or (ii) one and the same phylogenetically conserved basesequence at non-overlapping sequence regions, or (iii) one and the samephylogenetically conserved base sequence at overlapping sequenceregions, wherein (f) the set for an individual nucleic acid molecule,for a number of its individual nucleic acid molecules or for each of itsindividual nucleic acid molecules in each case comprises at least onefurther nucleic acid molecule that, in a region of at least 10successive nucleotides of their nucleotide chains, corresponds to lessthan 100% but to at least 80% of the base sequence, and wherein (g) theset of nucleic acid molecules does not comprise any degenerate nucleicacid molecules.
 3. Set of nucleic acid molecules by means of which, in aprocess for the detection of representatives of Salmonella entericasubsp. enterica, salamae, arizonae, diarizonae, houtenae, bongori andindica, all the representatives of those subspecies can be detected,which set of nucleic acid molecules is obtainable by (a) obtaining orderiving a first nucleic acid molecule (nucleic acid molecule 1) in amanner known per se using a nucleic acid isolate of a representative ofone of the mentioned Salmonella enterica subspecies, which first nucleicacid molecule is specifically suitable as primer or probe for thedetection of that representative or of further or all representatives ofthat one Salmonella enterica subspecies and possibly also ofrepresentatives of further Salmonella enterica subspecies, (b) obtainingor deriving a second nucleic acid molecule (nucleic acid molecule 2) ina manner known per se using a nucleic acid isolate of a differentrepresentative of one of the mentioned Salmonella enterica subspecies,which second nucleic acid molecule is specifically suitable as primer orprobe for the detection of that representative or of further or allrepresentatives of that different Salmonella enterica subspecies andpossibly also of representatives of others of the mentioned Salmonellaenterica subspecies, and (c) unless it is already possible to detect allthe representatives of the mentioned Salmonella enterica subspeciesusing the nucleic acid molecules obtainable or derivable according to(a) and (b), continuing to obtain or derive nucleic acid moleculesaccording to (a) and/or (b) until all the representatives of thementioned Salmonella enterica subspecies can be detected using theobtained or derived set of nucleic acid molecules, wherein (d) thenucleic acid isolates comprise or are phylogenetically conserved basesequences or regions of those base sequences, wherein (e) the individualnucleic acid molecules or some of the nucleic acid molecules hybridiseto (i) different phylogenetically conserved base sequences, or (ii) oneand the same phylogenetically conserved base sequence at non-overlappingsequence regions, or (iii) one and the same phylogenetically conservedbase sequence at overlapping regions, wherein (f) the set for anindividual nucleic acid molecule, for a number of its individual nucleicacid molecules or for each of its individual nucleic acid molecules ineach case comprises at least one further nucleic acid molecule that, ina region of at least 10 successive nucleotides of their nucleotidechains, corresponds to less than 100% but to at least 80% of the basesequence, and wherein (g) the set of nucleic acid molecules comprisesonly complementing primers.
 4. Set according to claim 1, 2 or 3,characterised in that the set for an individual nucleic acid molecule,for a number of its individual nucleic acid molecules or for each of itsindividual nucleic acid molecules in each case comprises at least onefurther nucleic acid molecule that, in a region of at least 10successive nucleotides of their nucleotide chains, differs from theother or further nucleic acid molecule in precisely one base position.5. Set according to any one of the preceding claims, characterised inthat it comprises one or more, but not exclusively, nucleic acidmolecules that are fragments of the SEQ ID NO 1 according to WO 95/00664 or of its complementary sequence.
 6. Set according to any one of thepreceding claims, characterised in that the individual nucleic acidmolecules hybridise to the same strand of nucleic acid isolates ofrepresentatives of Salmonella enterica subspecies that are beingsubjected to the process for their detection.
 7. Nucleic acid moleculethat belongs to a set according to any one of the preceding claims orthat can be used for such a set, characterised in that, in a region ofat least 10 successive nucleotides of its nucleotide chain, the sequenceof the nucleic acid molecule corresponds exactly to a sequence region ofat least one representative of Salmonella enterica subspecies accordingto any one of claims 1, 2 or 3, the sequence region comprising or beinga phylogenetically conserved base sequence or a region of that basesequence.
 8. Nucleic acid molecule according to claim 7, characterisedin that, in a region of at least 10 successive nucleotides of itsnucleotide chain, it is 100% or at least 80% identical to acorresponding number of successive nucleotides of one or more of thefollowing sequences or their complementary sequences:ATGGATCAGAATACGCCCCG SEQ ID NO:1 ATGGATCAGAATACACCCCG SEQ ID NO:2CAGAATACGCCCCGTTCGGC SEQ ID NO:3 CAGAATACACCCCGTTCGGC SEQ ID NO:4CAGAATACGCCCCGTTCAGC SEQ ID NO:5 CAACCTAACTTCTGCGCCAG SEQ ID NO:6CAACCTAACTTCTGCACCAG SEQ ID NO:7 CAACCTAACCTCTGCGCCAG SEQ ID NO:8CAACCTAACTTCTGCGGCAG SEQ ID NO:9 CAGCCTAACTTCTGCGCCAG SEQ ID NO:10


9. Nucleic acid molecule characterised in that, in respect of itsequence, it is homologous to a nucleic acid molecule according toeither claim 7 or claim 8 and, in at least 10 successive nucleotides ofits nucleotide chain, (i) is identical to a nucleic acid moleculeaccording to either claim 7 or claim 8, or (ii) differs from a nucleicacid molecule according to either claim 7 or claim 8 in not more thanone nucleotide, or (iii) differs from a nucleic acid molecule accordingto either claim 7 or claim 8 in not more than two nucleotides. 10.Nucleic acid molecule according to any one of claims 7 to 9,characterised in that it is from 10 to 250 nucleotides long, andpreferably from 15 to 30 nucleotides long.
 11. Nucleic acid moleculeaccording to any one of claims 7 to 10, characterised in that it issingle-stranded or has a complementary strand.
 12. Nucleic acid moleculeaccording to any one of claims 7 to 11, characterised in that it ispresent (i) as DNA, or (ii) as RNA corresponding to (i), or (iii) asPNA, the nucleic acid molecule where appropriate having been modified orlabelled in a manner known per se for analytical detection processes,especially detection processes based on hybridisation and/oramplification.
 13. Nucleic acid molecule according to claim 12,characterized in that it is a modified or labelled nucleic acid moleculein which up to 20% of the nucleotides of at least 10 successivenucleotides of its nucleotide chain are building blocks known per se asprobes and/or primers, especially nucleotides that do not occurnaturally in bacteria.
 14. Nucleic acid molecule according to claim 12or claim 13, characterized in that it is a modified or labelled oradditionally modified or labelled nucleic acid molecule that comprises,in a manner known per se for analytical detection processes, one or moreradioactive groups, coloured groups, fluorescent groups, groups forimmobilisation on a solid phase, groups for an indirect or directreaction, especially for an enzymatic reaction, preferably usingantibodies, antigens, enzymes and/or substances having an affinity forenzymes or enzyme complexes, and/or other modifying or modified groupsof nucleic-acid-like structure that are known per se.
 15. Kit foranalytical detection processes, for the detection of bacteria of theSalmonella genus, characterised by (i) a set of nucleic acid moleculesaccording to any one of claims 1 to 6, or (ii) one or more nucleic acidmolecules according to any one of claims 7 to
 14. 16. Kit according toclaim 15, characterised in that the set of nucleic acid molecules wasproduced synthetically and that it was produced in at least two separatesynthesis batches.
 17. Kit according to claim 16, characterised in thatthe kit does not comprise any degenerate nucleic acid molecules.
 18. Useof a set of nucleic acid molecules according to any one of claims 1 to6, of one or more nucleic acid molecules according to any one of claims7 to 14 or of a kit according to any one of claims 15 to 17 to detectthe presence or absence of bacteria belonging to representatives ofSalmonella enterica subspecies according to claim 1, 2 or
 3. 19. Useaccording to claim 18, characterised in that nucleic acid hybridisationand/or nucleic acid amplification is carried out.
 20. Use according toclaim 19, characterised in that a polymerase chain reaction (PCR) iscarried out as nucleic acid amplification.
 21. Use according to claim18, 19 or 20, characterised in that differences between the genomic DNAand/or RNA of the bacteria to be detected and of the bacteria that arenot to be detected are determined at at least one nucleotide position inthe region of a nucleic acid molecule according to any one of claims 7to 14 and representatives of a group of bacteria of the Salmonella genusare detected, especially representatives of Salmonella entericasubspecies according to claim 1, 2 or 3.